Helium-neon excitable reticulocyte dyes derivable from halolepidines

ABSTRACT

A method for synthesizing dyes excitable by a helium-neon laser (excitable at 633 nm) from hololepidines, e.g., 7-halolepidine, which dyes are suitable for detection and enumeration of reticulocytes in human blood samples. In another aspect, the invention provides a method for immunotyping phenocytes. The method is based on the phenomenon that when dyes of certain structure intercalate into DNA or RNA, the intensity of the dye increases. Dyes suitable for this invention can be described as having (a) a first heterocyclic moiety, (b) a second heterocyclic moiety, and (c) a linking group that connects the first and second heterocyclic moieties. Both the first and second heterocyclic moieties must contain at least two rings, preferably fused together. The dye is characterized by conjugation, whereby the first moiety is ethylenically conjugated to the second moiety.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The present invention relates to dyes that are suitable forstaining ribonucleic acid polymers (RNA) and deoxyribonucleic acidpolymers (DNA) and are particularly suitable for staining reticulocytes.The invention further relates to a fluorescent composition.

[0003] 2. Discussion of the Art

[0004] In many cases, there is a need to detect RNA or substancescontaining RNA. For example, a reticulocyte is a substance known tocontain RNA. Detection of reticulocytes in a blood sample and theenumeration of these reticulocytes are valuable to clinicians. Thereticulocyte count of a blood sample is an indicator of erythropoieticactivity, is an indicator of acute hemorrhage and hemolytic anemia, andis a measure of response to iron, vitamin B₁₂, and folic acid therapy.As is known in the art, reticulocytes are precursors of mature red bloodcells, and hence the term reticulocyte embraces the evolution anddevelopment of a mature red blood cell.

[0005] Detection and enumeration of reticulocytes in a blood sample havebeen carried out by both manual and automated methods by usingappropriate stains such as new methylene blue (NMB), brilliant cresylblue (BCB), acridine orange, and pyronin Y.

[0006] Vital staining with the dye new methylene blue is considered tobe the reference method for reticulocyte determinations. In use, thisdye precipitates RNA. The method is carried out manually and requirescounting large numbers of cells with a microscope (for example, 500 to1,000 cells). Consequently, the method is slow, tedious, and is subjectto errors.

[0007] New methylene blue is nonfluorescent and true precipitated RNA isoften difficult to differentiate from precipitated stain. New methyleneblue stains by combining with intracellular RNA molecules-to form acolored complex, which is visible under microscopic examination onaccount of its size and color. However, under certain conditions, thedye molecule itself can form complexes with other dye molecules. Thesedye/dye complexes are indistinguishable from dye/RNA complexes, with thepossible result that counts are inaccurate and/or false positives forthe specific cell type of interest are obtained. This problem is morelikely when the dye solution has not been filtered to removenon-specific dye/dye complexes that have formed.

[0008] Acridine orange has been used for staining reticulocytes by bothmanual and automated procedures. Acridine orange, which is fluorescent,also precipitates RNA. Consequently, the use of this dye preventsquantitative estimates of RNA content because of potential quenching, aphenomenon caused by dye molecules interfering with one another in theenergy transfer process. Under quenching conditions, the energy transferprocess results in no net fluorescence emission.

[0009] Age profiles of cells based on RNA content being proportional tofluorescence are not reliable. Age profile is the key information soughtto be derived in assays of blood cells. The function of the dye forstaining reticulocytes is primarily to determine the percentage ofimmature red blood cells in the general circulation. The information isneeded for determining the homeostasis of the blood cell formation,detection of blood cell related metabolic diseases, and the presence orabsence of anemic diseases. Acridine orange has a great affinity for theplastic tubing in flow cytometers, thereby resulting in increasedbackground, consequently requiring lengthy procedures for removing thedye from the flow cytometer tubing. In addition, cells stained byacridine orange are difficult to distinguish from the autofluorescentred cell peak. Finally, the reticulocyte count is usually lower thanthat obtained with new methylene blue. New methylene blue stains cellsby combining with the intracellular RNA to form an insoluble precipitatewithin the cells. A discrete blue pattern is formed upon theinteraction, thereby allowing for easy manual microscopic evaluation.Detection by means of acridine orange is performed on a flow cytometer.On account of the nature of the diffused pattern, it is difficult todifferentiate the specific staining from acridine orange to that ofnon-specific background. Consequently, if the background is high, thenet positive usually will be reduced and result in an artificially lowvalue, compared with the more discrete new methylene blue stainingmethod.

[0010] The use of pyronin Y requires prior fixation of the erythrocyteswith formalin, is cumbersome, time consuming, and generally yields poorresults. Moreover, pyronin Y has a very low quantum efficiency, leadingto a very low fluorescent signals.

[0011] Accordingly, there is a need for providing a dye better suitedfor staining reticulocytes so as to provide a procedure for accuratelydetermining reticulocytes in a blood sample.

[0012] A dye for staining reticulocytes preferably has the followingproperties:

[0013] 1. The dye should not fluoresce in the absence of RNA.

[0014] 2. The dye should have a good fluorescent quantum yield in thepresence of RNA.

[0015] 3. The dye must exhibit a certain level of water solubility andbe able to penetrate the membrane of cells containing RNA.

[0016] 4. The dye should preferably have an excitation peak at about 633nm.

[0017] U.S. Pat. No. 4,957,870 involves the detection of reticulocytes,RNA, and DNA in human blood samples using a dye having the followingstructure:

[0018] wherein

[0019] X represents O, S, Se or C(CH₃)₂;

[0020] R₁ represents an alkyl group having from 1 to 6 carbon atoms;

[0021] R₂ represents an alkyl group having from 1 to 6 carbon atoms;

[0022] R₃ represents fused benzene, alkyl group having from 1 to 6carbon atoms, methoxy, or is absent;

[0023] R₄ represents an alkyl group having from 1 to 6 carbon atoms,ethoxy, or is absent; and

[0024] n represents zero or an integer from 1 to 6.

SUMMARY OF THE INVENTION

[0025] In one aspect, this invention provides a method for synthesizingdyes excitable by a helium-neon laser (excitable at 633 nm). The dyesare preferably derived from heterocyclic compounds, e.g.,7-halolepidine. The dyes are suitable for the detection and enumerationof reticulocytes in human blood samples. In another aspect, thisinvention provides a method for signal amplification. The method isbased on the phenomenon that when dyes of certain structure intercalateinto DNA or RNA, the intensity of the dyes increases.

[0026] Dyes suitable for this invention can be described as having (a) afirst heterocyclic moiety, (b) a second heterocyclic moiety, and (c) alinking group that connects the first and second heterocyclic moieties.Both the first and second heterocyclic moieties must contain at leasttwo rings, preferably fused together. The dye is characterized byconjugation whereby the first moiety is ethylenically conjugated to thesecond moiety.

[0027] The general structure of dyes suitable for use in this inventionis shown below:

[0028] wherein:

[0029] R¹, R², R³, R⁴ independently represent a member selected from thegroup consisting of hydrogen, halogen, cyano, alkyl, aryl, alkaryl,aralkyl, or R¹ and R² taken together, or R² and R³ taken together, or R³and R⁴ taken together represent one or more rings;

[0030] R⁵ represents an alkyl group or an aryl group;

[0031] R⁶, R⁷, R⁸, R⁹ independently represent a member selected from thegroup consisting of hydrogen, halogen, cyano, alkyl, aryl, alkaryl,aralkyl, or R⁶ and R⁷ taken together, or R⁷ and R⁸ taken together, or R⁸and R⁹ taken together represent one or more rings, provided that atleast one of R⁶, R⁷,

[0032] R⁸, R⁹ represents halogen;

[0033] R¹⁰ represents an alkyl group or an aryl group;

[0034] R¹¹, R¹² independently represent a member selected from the groupconsisting of hydrogen, halogen, cyano, alkyl, aryl, alkaryl, aralkyl,or R¹¹ and R¹² taken together represent one or more rings;

[0035] R¹³ represents hydrogen or an alkyl group;

[0036] Y represents S, O, C, or Se, provided that Y is not N;

[0037] n represents a number from 0 to 3; and

[0038] X⁻ represents a counter ion.

[0039] The ring of the first moiety that is attached to the linkinggroup can be a five-membered or six-membered ring. The ring of thesecond moiety that is attached to the linking group can be afive-membered or six-membered ring.

[0040] R⁵ and R¹⁰ can be the same or different. Preferably, R⁵ and R¹⁰represent an alkyl group having from 1 to 20 carbon atoms and from 0 to6 heteroatoms, preferably from 1 to 10 carbon atoms and from 0 to 3heteroatoms. R⁵ and R¹⁰ can be a straight chain, branched chain, orcyclic group. If R⁵ of R¹⁰ is an alkyl group, the carbon atoms of R⁵ orR¹⁰ can contain substitutents other than hydrogen atoms. R⁵ and R¹⁰ canalso be (a) aryl groups, preferably having no more than five rings, morepreferably no more than three rings, most preferably no more than onering, or (b) alkenyl groups, preferably having from 2 to 20 carbonatoms, more preferably from 2 to 10 carbon atoms. If R⁵ or R¹⁰ is anaryl or an alkenyl group, the aryl or alkenyl groups of R⁵ or R¹⁰ cancontain substitutents other than hydrogen atoms. R⁵ and R¹⁰ can beheteroaryl group groups, wherein heteroatoms can be selected from thegroup consisting of nitrogen, sulfur, oxygen, and selenium.

[0041] The identities of substituents for R⁵ and R¹⁰ are not critical,but they should be selected so as not to adversely affect the absorptioncharacteristics of the dyes.

[0042] With respect to the value of n, there must be sufficient carbonatoms in the linking group to provide the desired absorptioncharacteristics of the dye.

[0043] When R⁶, R⁷, R⁸, or R⁹ is a halogen, the halogen is preferablyselected from the group consisting of F, Cl, and I.

[0044] If R¹, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹¹, R¹², or R¹³ is alkyl, itpreferably contains 1 to 20, more preferably 1 to 10, and mostpreferably 1 to 6 carbon atoms. If R¹, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹¹,R¹², or R¹³ is aryl, it prefably contains no more than five rings, morepreferably no more than three rings, and most preferably no more thanone ring. If R¹, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹¹, R¹², or R¹³ is alkylor aryl, it can contain substituents other than hydrogen. If R¹ and R²taken together, or R² and R³ taken together, or R³ and R⁴ takentogether, or R⁶ and R⁷ taken together, or R⁷ and R⁸ taken together, orR⁸ and R⁹ taken together, or R¹¹ and R¹² taken together form one or morerings, the rings can be aromatic or non-aromatic. The aromatic rings canbe carbocyclic or heteroaromatic, wherein the hetroatoms are selectedfrom the group consisting of nitrogen, sulfur, oxygen, and selenium.Preferably, ring structures formed by the foregoing combinations containno more than five rings, preferably no more than three rings, and mostpreferably no more than one ring.

[0045] Dyes suitable for this invention exhibit very low background,narrow emission bands, and excellent enhancement when DNA or RNA ispresent. The dyes are stable to oxygen, moisture, and heat but areslowly decolored when exposed to light.

[0046] The dyes of this invention can be used as a signal generatingagent for the detection of DNA or RNA. The dyes of this invention aresufficiently sensitive such that they can be used for the detection ofreticulocytes in human blood samples on a flow cytometer usinghelium-neon (He—Ne) laser as the light source. Helium-neon lasers aremuch less expensive than argon lasers.

[0047] As mentioned previously, the dyes of this invention exhibit muchhigher enhancement, lower background, and smaller emission bandwidththan dyes used in the prior art. The low background and smaller emissionbandwidth enables the dyes to be used for high resolution analyses ofwhole blood and in multiplexing assays.

DETAILED DESCRIPTION

[0048] Dyes suitable for this invention can be described as having afirst heterocyclic moiety, a second heterocyclic moiety, and a linkinggroup that connects the first and second heterocyclic moieties. Both thefirst and second heterocyclic moieties must contain at least two rings,preferably fused together. The dye is characterized by conjugationwhereby the first moiety is ethylenically conjugated to the secondmoiety.

[0049] The general structure of dyes suitable for use in this inventionis shown below:

[0050] wherein:

[0051] R¹, R², R³, R⁴ independently represent a member selected from thegroup consisting of hydrogen, halogen, cyano, alkyl, aryl, alkaryl,aralkyl, or R¹ and R² taken together, or R² and R³ taken together, or R³and R⁴ taken together represent one or more rings;

[0052] R⁵ represents an alkyl group or an aryl group;

[0053] R⁶, R⁷, R⁸, R⁹ independently represent a member selected from thegroup consisting of hydrogen, halogen, cyano, alkyl, aryl, alkaryl,aralkyl, or R⁶ and R⁷ taken together, or R⁷ and R⁸ taken together, or R⁸and R⁹ taken together represent one or more rings, provided that atleast one of R⁶, R⁷, R⁸, R⁹ represents halogen;

[0054] R¹⁰ represents an alkyl group or an aryl group;

[0055] R¹¹, R¹² independently represent a member selected from the groupconsisting of hydrogen, halogen, cyano, alkyl, aryl, alkaryl, aralkyl,or R¹¹ and R¹² taken together represent one or more rings;

[0056] R¹³ represents hydrogen or an alkyl group;

[0057] Y represents S, O, C, or Se, provided that Y is not N;

[0058] n represents a number from 0 to 3; and

[0059] X⁻ represents a counter ion.

[0060] The ring of the first moiety that is attached to the linkinggroup can be a five-membered or six-membered ring. The ring of thesecond moiety that is attached to the linking group can be afive-membered or six-membered ring.

[0061] R⁵ and R¹⁰ can be the same or different. Preferably, R⁵ and R¹⁰represent an alkyl group having from 1 to 20 carbon atoms and from 0 to6 heteroatoms, preferably from 1 to 10 carbon atoms and from 0 to 3heteroatoms. R⁵ and R¹⁰ can be a straight chain, branched chain, orcyclic group. The alkyl groups of R⁵ and R¹⁰ can contain substitutentsother than hydrogen atoms. R⁵ and R¹⁰ can also be (a) aryl groups,preferably having no more than five rings, more preferably no more thanthree rings, most preferably no more than one ring, or (b) alkenylgroups, preferably having from 2 to 20 carbon atoms, preferably from 2to 10 carbon atoms. The aryl or alkenyl groups of R⁵ and R¹⁰ can containsubstitutents other than hydrogen atoms.

[0062] The identities of substituents for R⁵ and R¹⁰ are not critical,but they should be selected so as not to adversely affect the absorptioncharacteristics of the dyes.

[0063] R⁵ and R¹⁰ can be heteroaryl group groups, wherein heteroatomscan be selected from the group consisting of nitrogen, sulfur, oxygen,and selenium.

[0064] With respect to the value of n, there must be enough carbon atomsin the linking group to provide the desired absorption characteristicsof teh dye.

[0065] When R⁶, R⁷, R⁸, or R⁹ is a halogen, the halogen is preferablyselected from the group consisting of F, Cl, and I.

[0066] If R¹, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹¹, R¹², or R¹³ is alkyl, itpreferably contains 1 to 20, more preferably 1 to 10, and mostpreferably 1 to 6 carbon atoms. If R¹, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹¹,R¹², or R¹³ is aryl, it prefably contains no more than five rings, morepreferably no more than three rings, and most preferably no more thanone ring. If R¹, R², R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹¹, R¹², or R¹³ is alkylor aryl, it can contain substituents other than hydrogen. If R¹ and R²taken together, or R² and R³ taken together, or R³ and R⁴ takentogether, or R⁶and R⁷ taken together, or R⁷ and R⁸ taken together, or R⁸and R⁹ taken together, or R¹¹ and R¹² taken together form one or morerings, the rings can be aromatic or non-aromatic. The aromatic rings canbe carbocyclic or heteroaromatic, wherein the hetroatoms are selectedfrom the group consisting of nitrogen, sulfur, oxygen, and selenium.Preferably, ring structures formed by the foregoing combinations containno more than five rings, preferably no more than three rings, and mostpreferably no more than one ring.

[0067] X⁻ represents a counter ion, preferably acetate.

[0068] The dyes are preferably soluble in water and stable underconditions of use, such as, for example, in a flow cytometer. The dyesare capable of being linked to a molecule, e.g., a protein or a polymer,through the moiety R⁵, the moiety R¹⁰, or another moiety.

[0069] When not bound to a nucleic acid, the dyes of the inventionexhibit a strong absorption peak in the range of from about 600 nm toabout 630 nm; however, in the unbound state, the dye does not provideeither a detectable excitation or emission peak. When the dyes stain theRNA in the reticulocytes, the optical properties of the dye changedramatically. In particular, the absorption curve shifts to a longerwavelength, and the dye exhibits strong fluorescence. For a typical dyeuseful in this invention, the excitation maximum is at about 633 nm, andthe emission maximum is at about 670 nm, giving a Stokes shift of about40 nm. As a result of the excitation peak of the bound dye being in theorder of about 633 nm, the light source for use with the automatic flowcytometer may be a helium-neon laser, which has strong emission at 633nm. The lack of fluorescence of the dye when not bound to nucleic acidprovides low backgrounds and allows an operator to select a fluorescentthreshold (or “gate”) for a flow cytometer by simply running anunstained control. Although excitation may be effected at otherwavelengths, the reticulocytes stained with the dyes described hereinare preferably excited at a wavelength of from about 630 nm to about 670nm Representative examples of dyes suitable for use in this inventionhave the following structural formulas. In the following structures,“Ph” represents the phenyl group, “Ar” represents an aryl group, asingle straight line represents —CH₃, and a broken line represents—CH₂CH₃.

[0070] The following table sets forth the absorption maximum innanometers for several dyes suitable for use in this invention. TABLE IDye number Absorption maximum (nm) 2 628 5 638 6 632 7 638 8 638 9 624

[0071] Dyes suitable for use in this invention can be prepared by

[0072] (1) reacting a heterocyclic compound containing at least twofused rings with acetic anhydride and N,N′-diphenylformamidine or itshigher homologs to form a chain-extended intermediate;

[0073] (2) reacting the aforementioned chain-extended intermediate witha methylated halolepidine in the presence of a tertiary alkyl aminecatalyst under reflux conditions to form the dye.

[0074] The resultant dye can then be recovered by precipitation,typically by diethylether. Alternatively, dyes suitable for thisinvention can be prapred by

[0075] (1) reacting a heterocyclic compound containing at least twofused rings and an activated methyl group with acetic anhydride andN,N′-diphenylformamidine or its higher homologs to form a chain-extendedintermediate;

[0076] (2) reacting the aforementioned chain-extended intermediate witha halolepidine in the presence of a tertiary alkyl amine catalyst underreflux conditions to form the dye.

[0077] The resultant dye can then be recovered by precipitation,typically by diethylether. Representative examples of compoundscontaining at least two fused rings include 2-methyl-N-alkylbenzothiazolium iodide, 2-methyl-N-alkyl benzoxazolium iodide,2-methyl-N-alkyl naphthoxazolium iodide, and 2-methyl-N-alkylnapthothiazolium iodide. Representative examples of N-methylatedhalolepidines 7-chlorolepidine, 7-fluorolepidine, and 7-bromolepidine.

[0078] Dyes suitable for use in this invention can be prepared accordingto the following scheme of synthesis. In the following scheme ofsynthesis, “Ph” represents the phenyl group and “Ac” represents the acylgroup, and “X” represents the halo group.

[0079] In the foregoing scheme of synthesis, representative sets of thesubstituents R and X include the following: TABLE II Dye R X 2 —CH₃ —F 5—CH₃ —Br 6 —CH₃ —Cl 7 —CH₂CH₃ —Cl 8 —CH₂CH₃ —Cl 9 —CH₃ —Cl

[0080] In accordance with the present invention, the dyes of theinvention may be employed in the form of an aqueous solution whenstaining reticulocytes in a blood sample, and, in particular, in theform of an isotonic saline solution. The saline solution may contain aminor amount of methanol. The blood sample, which may be whole blood ora blood fraction, is stained with the dye by mixing the blood samplewith the solution comprising the dye. It has been found that by usingthe dyes desribed herein as the staining medium, it is possible todetect and enumerate reticulocytes in a whole blood sample.

[0081] Because the dye must permeate the reticulocyte, it should haveexcellent cell permeation properties. The dyes suitable for thisinvention do not precipitate RNA, and as a result, the stainedreticulocyte cells maintain a relatively homogeneous distribution ofintracellular RNA, whereby it is possible to designate a threshold valuedemarcating the distinction between a reticulocyte and a mature redcell. This characteristic provides the physician with additionalinformation beyond the reticulocyte count in that RNA content is afunction at the age of the reticulocytes. Accordingly, by using a dyedescribed herein, a clinician has the ability to perform reticulocyteage profiles as well as simple reticulocyte counts. The use of dyesdescribed herein for staining reticulocytes in a blood sample offers thefurther advantage that the fluorescent signals from the stainedreticulocytes are readily distinguishable from those of the matureerythrocytes, which contain no RNA or DNA. For this reason, results canbe directly read in a flow cytometer without extensive datamanipulation.

[0082] Although reticulocytes and RNA or DNA stained with a dye of theinvention are preferably enumerated in an automatic flow cytometer, theycan also be counted by a manual procedure or automated microscopy.

[0083] The present invention is not limited to-the use of any particularflow cytometer. Thus, for example, stained reticulocytes may be detectedand enumerated in an automated flow cytometer or a semi-automated flowcytometer or a manual flow cytometer. In using automated flowcytometers, fluorescent gates are set by use of an unstained controlsample, and the fluorescent gates are then used on the stained sample.

[0084] The dyes described herein can be used directly to stainreticulocytes. They need not be attached to an antibody or the like toform a conjugate. Staining can be brought about by an intercalationmechanism, whereby intercalation of the dye with RNA or DNA of thereticulocyte causes a blue shift or a red shift in excitation oremission wavelength.

[0085] Alternatively, the dyes described herein can be used in the formof a conjugate. One application of using a conjugate comprising a dyedescribed herein is in a flow cytometry application that employs afluorescent conjugate or multiple fluorescent conjugates to detect cellscontained in a test sample. An example of a flow cytometer is theFluorescence Activated Cell Sorter (FACScan) manufactured by Becton,Dickinson & Co, Franklin Lakes, N.J. In general, an imaging systemcontains an excitation source and a detection device. The excitationsource excites the fluorescent signal generating group associated withthe conjugate and the detection device detects the signal emitted fromthe excited signal generating group.

[0086] In a typical imaging system analysis, a test sample is incubatedwith a fluorescent conjugate, which specifically binds certain cellsthat may be present in the test sample. The incubation takes place for atime and at a temperature conducive for the binding of the conjugate tospecific cell populations contained in the sample. The cells bound withthe conjugate are commonly referred to as being stained and the stainingprocedure can be executed multiple times, sequentially or at the sametime, with multiple conjugates, which emit signals of varyingwavelengths. After the staining procedure is complete, the sample can beanalyzed using a flow cytometer.

[0087] In an alternative embodiment, a conjugate comprising the dyesdescribed herein can be adapted for use in conventional solid phaseimmunoassays such as, for example, a sandwich type immunoassay. Asandwich type immunoassay typically involves contacting a test samplesuspected of containing an analyte with a substantially solid inertplastic, latex or glass bead or microparticle, or other support materialwhich has been coated with a specific binding member that forms abinding pair with the analyte. The binding member-coated supportmaterial is commonly referred to as a “capture reagent”. After theanalyte is bound to the support material, the remaining test sample isremoved from the support material. The support material, to which theanalyte is bound, is treated with a conjugate, which generally comprisesa second binding member labeled with a signal-generating group. Theconjugate becomes bound to the analyte, which is bound to the supportmaterial. The combination of support material having the first bindingmember, the analyte, and the conjugate bound thereon is separated fromany unbound conjugate, typically with one or more wash steps. The signalgenerated by the signal generating group, through appropriateexcitation, can then be observed visually, or more preferably by aninstrument, to indicate the presence or amount of an analyte in a testsample. It will be understood, of course, that the order and number ofthe steps employed to perform such assays are not intended to limit theinvention described herein.

[0088] The analyte detected by such an immunoassay can be the product orproducts of an amplification reaction. Accordingly, the analytes cancomprise nucleic acid sequences or can be otherwise the products of ahybridization reaction such as LCR, which is described in EuropeanPatent Applications EP-A-320 308 and EP-A-439 182, and PCR, which isdescribed in U.S. Pat. Nos. 4,683,202 and 4,683,195, all of which areincorporated herein by reference. In cases where the analytes comprise,for example, LCR or PCR reaction products or sequences, the sequencescan comprise or be modified to comprise a binding member that forms abinding pair with an indicator reagent and a binding member that forms abinding pair with a capture reagent.

[0089] The use of reticulocytes stained with the dyes described hereinin a flow cytometer is particularly advantageous in that there are lowfluorescent backgrounds, and fluorescent gates may be easily selected byuse of an unstained control. Moreover, because there is no precipitationof intracellular reticulocyte RNA, whereby the cells need not be fixed.In addition, the relationship between the fluorescent signal and theindividual reticulocytes provides information as to the age of thereticulocytes.

[0090] Still another advantage of the prevent invention is thatreticulocytes stained with the dyes descibed herein can be used in anautomated flow cytometer having lower light intensity, e.g., one may usea helium-neon laser as opposed to an argon laser.

[0091] The following examples illustrate various features of the presentinvention but is not intended to in any way limit the scope of theinvention as set forth in the claims.

EXAMPLE I Preparation of 7-chlorolepidine

[0092] To a mixture containing 3-chloroaniline (1.59 g), ferric chloridehexahydrate (5.4 g), zinc chloride (0.2 g), ethanol (20 ml of 95%aqueous solution) preheated to 60° C. was added 1,3,3-trimethoxybutane(1.48 g). The resulting mixture was refluxed for two hours and allowedto stand overnight. The volatile matrials were then removed in vacuo andthe residue rendered basic with 10% aqueous sodium hydroxide. Theresulting mixture was partitioned between water and diethyl ether (3times). The combined ether layer was dried over magnesium sulfate.Rotary evaporation of the ether solution gave a dark liquid. The liquidwas added to a silica gel column and eluted with hexane/ethyl acetate(3:1) to give the desired product as tan crystals.

Preparation of 7-chloro-1-methyllepidinium iodide

[0093] A portion of the 7-chlorolepidine (80 mg) prepared as above wasmethylated by heating with CH₃I (0.5 ml) in CH₃CN (1 ml) at reflux fortwo hours. The mixture was treated with diethyl ether, followed bycentrifugation, to give a yellow powder.

Activation of 3-ethyl-2-methylbenzothiazolium iodide

[0094] A mixture of 3-ethyl-2-methylbenzothiazolium iodide (350 mg) andN,N′-diphenylformamidine (420 mg) in acetic anhydride (10 ml) was heatedto 120° C. for 30 minutes. Diethyl ether was added and the suspensionwas centrifuged. The supernatant was decanted and the precipitate washedwith more diethyl ether and dried.

Preparation of Dye 7

[0095] To the activated 3-ethyl-2-methylbenzothiazolium derivativeobtained above (4.5 mg) was added the 7-chloro-1-methyllepidinium saltobtained above (3.8 mg) in chloroform (250 μL) to form a mixture. Thentriethylamine (50 μL) was added to the mixture. The resulting mixturewas stirred at reflux for 30 minutes, during which time a dark bluesolution was obtained. Diethyl ether was added to the solution and theresultant suspension was centrifuged. The supernatant was discarded andthe precipitate resuspended in diethyl ether and centrifuged (2 times).The dark powder absorbed maximally at 638 nm in methanol solution.

EXAMPLE II Preparation of Dye 8

[0096] The procedure described for the preparation of Dye 7 wasfollowed, with the exception that 2,3-dimethylbenzothiazolium iodide wassubstituted for 3-ethyl-2-methylbenzothiazolium iodide in EXAMPLE I. Thedye also had an absorption maximum at 638 nm in methanol.

EXAMPLE III Preparation of Dye 9

[0097] The procedure described for the preparation of Dye 7 wasfollowed, with the exception that 2,3-dimethylnaphthoxazolium iodide wassubstituted for 3-ethyl-2-methylbenzothiazolium iodide in EXAMPLE I. Thedye had an absorption maximum of 624 nm in methanol.

EXAMPLE IV Preparation of Dye 2

[0098] The procedure described for the preparation of Dye 7 wasfollowed, with the exception that 3-fluoroaniline (1.84 g) wassubstituted for 3-chloroaniline in EXAMPLE I. The dye had an absorptionmaximum of 628 nm in methanol.

EXAMPLE V Preparation of Dye 5

[0099] The procedure described for the preparation of Dye 7 wasfollowed, with the exception that 3-bromoaniline (2.15 g) wassubstituted for 3-chloroaniline in EXAMPLE I. The dye had an absorptionmaximum at 638 nm in methanol.

EXAMPLE VI Preparation of Dye 6

[0100] The procedure described for the preparation of Dye 7 wasfollowed, with the exception that 1,1,2,3-tetramethyl-1H-benz(e)indoliumiodide was substituted for 3-ethyl-2-methylbenzothiazolium iodide inEXAMPLE I. The dye had an absorption maximum at 623 nm in methanol.

EXAMPLE VII

[0101] Fluorescence was determined by dissolving the dye in a buffer(“CD4000 Retic Buffer”) at neutral pH and at a final concentration of0.5 μg/ml of test solution. The “CD4000 Retic Buffer” contained thefollowing ingredients in the amounts indicated: Ingredient AmountImidazole 3.40 g HCl, 1 N 23.5 mL NaCl 6.80 g “BIGCHAP” (N,N-bis[3-D-0.05 g Gluconamidopropyl]cholamide) “PROCLIN 300” (mixture of 5-chloro-0.315 g 2-methyl-isothiazolone and 2-methyl- 3(2H)-isothiazolone)Deionized water to 1.0 liter

[0102] The tests were run in a flow cytometer with a helium-neon laserlight source. The helium-neon laser provided light at a wavelength of630 nm. The optical system was a conventional optical system. Theresults are set forth in TABLE III. TABLE III Dye of U.S. Pat. No.4,957,870 Dye 2 Dye 7 Emission 651.0 646.4 655.6 wavelength - dye only(nm) Emission 661.0 662.6 671.2 wavelength with RNA (nm) Emission 652.6654.8 661.8 wavelength with DNA (nm) Intensity - dye only 1.0 1.0 1.0Enhancement with 55.10 91.80 39.1 RNA Enhancement with 54.10 76.10 50.4DNA Bandwidth 40 45 Bandwidth with RNA 40 36 Bandwidth with 40 35 DNA

[0103] From the data in TABLE III, it can be seen that Dye 10 of thepresent invention provide greater signal enhancement with DNA and withRNA than does the dye of U.S. Pat. No. 4,957,870.

[0104] Various modifications and alterations of this invention willbecome apparent to those skilled in the art without departing from thescope and spirit of this invention, and it should be understood thatthis invention is not to be unduly limited to the illustrativeembodiments set forth herein.

What is claimed is:
 1. A dye having (a) a first heterocyclic moiety, (b)a second heterocyclic moiety, and (c) a linking group that connects thefirst and second heterocyclic moieties, said first and secondheterocyclic moieties must having at least two rings, said heterocyclicmoieties being characterized by conjugation whereby the first moiety isethylenically conjugated to the second moiety.
 2. The compound of claim1, wherein the ring of the first moiety that is attached to the linkinggroup can be a five-membered or six-membered ring.
 3. The compound ofclaim 1, wherein the ring of the second moiety that is attached to thelinking group can be a five-membered or six-membered ring.
 4. A compoundhaving the structure

wherein: R¹, R², R³, R⁴ independently represent a member selected fromthe group consisting of hydrogen, halogen, cyano, alkyl, aryl, alkaryl,aralkyl, or R¹ and R² taken together, or R² and R³ taken together, or R³and R⁴ taken together represent one or more rings; R⁵ represents analkyl group or an aryl group; R⁶, R⁷, R⁸, R⁹ independently represent amember selected from the group consisting of hydrogen, halogen, cyano,alkyl, aryl, alkaryl, aralkyl, or R⁶ and R⁷ taken together, or R⁷ and R⁸taken together, or R⁸ and R⁹ taken together represent one or more rings,provided that at least one of R⁶, R⁷, R⁸, R⁹ represents halogen; R¹⁰represents an alkyl group or an aryl group; R¹¹, R¹² independentlyrepresent a member selected from the group consisting of hydrogen,halogen, cyano, alkyl, aryl, alkaryl, aralkyl, or R¹¹ and R¹² takentogether represent one or more rings; R¹³ represents hydrogen or analkyl group; Y represents S, O, C, or Se, provided that Y is not N; nrepresents a number from 0 to 3; and X⁻ represents a counter ion.
 5. Thecompound of claim 4, wherein R⁵ represents an alkyl group having from 1to 20 carbon atoms.
 6. The compound of claim 4, wherein R¹⁰ representsan alkyl group having from 1 to 20 carbon atoms.
 7. The compound ofclaim 4, wherein R⁵ represents an aryl group, preferably having no morethan five rings, more preferably no more than three rings, mostpreferably no more than one ring.
 8. The compound of claim 4, whereinR¹⁰ represents an aryl group, preferably having no more than five rings,more preferably no more than three rings, most preferably no more thanone ring.
 9. The compound of claim 4, wherein R⁵ represents an alkenylgroup, preferably having from 2 to 20 carbon atoms, more preferably from2 to 10 carbon atoms.
 10. The compound of claim 4, wherein R¹⁰represents an alkenyl group, preferably having from 2 to 20 carbonatoms, more preferably from 2 to 10 carbon atoms.
 11. The compound ofclaim 4, wherein R⁵ represents a heteroaryl group, wherein heteroatomscan be selected from the group consisting of nitrogen, sulfur, oxygen,and selenium.
 12. The compound of claim 4, wherein R¹⁰ represents aheteroaryl group, wherein heteroatoms can be selected from the groupconsisting of nitrogen, sulfur, oxygen, and selenium.
 13. The compoundof claim 4, wherein at least one of R⁶, R⁷, R⁸, or R⁹ is selected fromthe group consisting of F, Cl, and I.
 14. A method for preparing thecompound of claim 4 comprising the step of reacting an alkylatedhalolepidine with the reaction product of N,N′-diphenylformadidine,acetic anhydride, and a heterocyclic compound containing at least twofused rings in the presence of a tertiary alkyl amine catalyst underreflux conditions.
 15. The method of claim 14, wherein said alkylatedhalolepidine is the reaction product of haloaniline and trialkoxyalkane.16. The method of claim 14, wherein said heterocyclic compoundcontaining at least two fused rings is selected from the groupconsisting of 2-methyl-N-alkyl benzothiazolium iodide, 2-methyl-N-alkylbenzoxazolium iodide, 2-methyl-N-alkyl naphthoxazolium iodide, and2-methyl-N-alkyl naphthothiazolium iodide.
 17. A method for preparingthe compound of claim 4 comprising the step of reacting a halolepidinewith a heterocyclic compound containing at least two fused rings and anactivated methyl group with acetic anhydride andN,N′-diphenylformamidine in the presence of a tertiary alkyl aminecatalyst under reflux conditions.
 18. The method of claim 17, whereinsaid alkylated halolepidine is the reaction product of haloaniline andtrialkoxyalkane.
 19. The method of claim 17, wherein wherein saidheterocyclic compound containing at least two fused rings is selectedfrom the group consisting of 2-methyl-N-alkyl benzothiazolium iodide,2-methyl-N-alkyl benzoxazolium iodide, 2-methyl-N-alkyl naphthoxazoliumiodide, and 2-methyl-N-alkyl naphthothiazolium iodide.
 20. A method ofimmunotyping phenocytes comprising the steps of: a. providing a testsample; b. isolating cells from said test sample; c. introducing alysing to said test sample; d. adding the dye of claim 1 to said testsample; e. measuring fluorescence of said test sample.
 21. The method ofclaim 20, wherein said method of immunotyping phenocytes is flowcytometry.
 22. The method of claim 21, wherein said method ofimmunotyping phenocytes is image analysis.